The present invention relates to novel affinity ligands, their preparation and attachment to matrices which may consist of solid, semi-solid, particulate or colloidal materials, or soluble polymers. The invention furthermore relates to these novel affinity ligand-matrix conjugates and the preparation and use thereof in the binding and removal of endotoxin from various fluids such as water, aqueous solutions, body fluids, blood, plasma, solutions of pharmaceutical products, proteins and other compounds of biological origin.
Endotoxins are lipopolysaccharides found in the outermost membrane of Gram-negative bacteria, particularly pathogeneic bacteria of the class Enterobacteriaceae, Neisseriaceae and Chlamydiaceae. Endotoxins comprise lipid A attached to a polysaccharide of variable structure dependent upon its biological origin. The polysaccharide component of Enterobacteriaceae endotoxin is characterised by an O-specific chain region and a core region. The O-specific region comprises up to 50 repeating oligosaccharide units that contain as many as 8 different sugar residues. O-specific chains exhibit large structural diversity from species to species whereas the core region, divided into the outer core and inner core regions, is less variable. The inner core region is characterised by the presence of unusual sugar residues such as heptose and 2-keto-3deoxyoctonic acid (KDO) which are frequently substituted with phosphate or phosphate derivatives. Also attached to the inner core region, lipid A is a conserved biphosphorylated glucosamine disaccharide which is acylated by 4 saturated primary acyl groups of which 2 carry secondary saturated acyl groups. The combination of hydrophobic lipid A tails with the hydrophilic and anionic polysaccharide unit provides endotoxin with amphipathic properties.
Endotoxin released from the cell wall of Gram-negative bacteria is considered to be the primary cause of the many pathiphysiological occurrences that accompany Gram-negative septicaemia. Endotoxin at pg/ml concentrations in blood triggers the release of a variety of cytokines, including interleukins and TNF. Over stimulation of the immune system by endotoxin leads to a massive release of cytokines which ultimately results in metabolic breakdown and septic shock. During septic shock, the complement and coagulation cascades become activated and vascular permeability increases. This can lead to disseminated intravascular coagulation and multiple organ failure, often with fatal consequences. Septic shock often develops because of the lack of an initial response to infection allowing the level of blood-borne endotoxin to reach critical levels.
In addition to the obvious risk presented by the presence of live Gram negative bacteria or cell wall debris in parenteral pharmaceutical products, the presence of free endotoxin in pharmaceutical preparations is also a major concern. Because endotoxin is such a potent immune stimulator, very low concentrations may cause toxic reactions including pyrogenic effects. Endotoxin is a relatively stable molecule which is not inactivated by routine autoclaving or treatment with organic solvents. Exposure to concentrated sodium hydroxide or prolonged high temperature (250xc2x0 C.) will inactivate endotoxin, though such methods are not appropriate for most biological products. Furthermore, maintenance of complete sterility throughout the manufacture of bio-therapeutics is problematic. Consequently, the highly efficient capture and removal of endotoxin from parenteral pharmaceuticals is very desirable, particularly in situations where endotoxin is known to associate with components of the therapeutic formulation.
A variety of techniques have been used to remove endotoxin from aqueous solutions including ultrafiltration, charcoal adsorption, cation-exchange chromatography, and a variety of immobilised affinity ligands including polymyxin B and endotoxin binding protein. All of these techniques exhibit significant shortcomings, particularly in the case of endotoxin removal from high molecular weight compounds such as therapeutic proteins. Ultrafiltration can only be used to remove endotoxin from low molecular weight compounds whereas charcoal adsorption tends to promote the binding of most organic compounds. Cation-exchange chromatography is effective in removing endotoxin from water but less effective for protein containing solutions, particularly proteins with acidic isoelectric points. Polymyxin B, a cyclic polypeptide antibiotic, is too toxic to allow its use for the purification of therapeutic products whereas endotoxin binding protein is too expensive for commercial applications.
Immobilised cationic amino acids (histidine, lysine and arginine) have also been used for endotoxin removal (Tosa, T. et al., Molecular Interactions in Bioseparations, Ed. Ngo, T. T., Plenum Press, New York, pp. 323-332, 1993; Lawden, K. H. et al, Bacterial Endotoxins: Lipopolysaccharides From Genes to Therapy, Wiley-Liss Inc., pp. 443-452, 1995). Such materials have been prepared by direct attachment of amino acids to epoxy-activated chromatographic matrices. In the case of Pyrosep(trademark), a commercially available material manufactured by Tanabe Seiyaku. Company Limited, Osaka, Japan, a single histidine group is immobilised to a support matrix by a hexanediamine spacer arm. Again, such materials are adequate for removal of endotoxin from water or solutions of low molecular weight compounds, but their performance is compromised in the presence of salt ( greater than 50 mM) or proteins which have an affinity for endotoxin. Consequently, none of the existing methods of endotoxin removal are suited to the elimination of endotoxin from bio-therapeutic compounds intended for parenteral administration. This is especially true for protein therapeutics where no single effective and safe method of endotoxin removal exists.
Removal of endotoxin from blood or plasma may provide an effective approach to the management of septic shock, particularly if applied at the early stages of infection or prophylactically in situations where an increased risk of septic shock is anticipated (e.g. major bowel or liver surgery). Several studies have been reported as to the use of monocolonal antibodies directed against endotoxin or cytokines released in the initial phase of the shock reaction. However, most of these approaches have been found to be ineffective (Siegel, J. P., Drug Information Journal, 30, pp. 567-572, 1996). In contrast, extracorporeal extraction of endotoxin from whole blood has been accomplished by use of fibre-immobilised polymyxin B (Aoki, H. et al., Nippon Geka Gakkai Zasshi (Japan), 94, pp. 775-780, 1993), though concerns over potential toxicity of polymyxin B lechates remain. Consequently, affinity adsorbents incorporating endotoxin binding ligands which have high affinity for endotoxin and low toxicity may also be beneficial for the management of sepsis.
Immobilised amino acids have also been investigated as potential endotoxin removal agents but such materials bind endotoxin weakly and non-specifically and are of limited value in the extraction of endotoxin from biological fluids and solutions of biological compounds. Triazine-based compounds have been reported which bind selectively to proteins; however, such ligands are not applicable to the isolation of endotoxin.
This invention relates to the discovery of synthetic affinity ligand structures which bind selectively to endotoxin. A generic group of novel affinity ligands have been found which exhibit high affinity for endotoxin and are generally applicable to the isolation of endotoxin from a variety of sources.
A feature of the present invention is the provision of a general tool for the removal of endotoxin contamination from biological materials. Endotoxin binds exceedingly tightly to affinity ligand-matrix conjugates of the invention. This feature enables highly efficient extraction of endotoxin from water and aqueous solutions providing a means of generating pyrogen-free water or pyrogen-free solutions. Affinity ligand-matrix conjugates of the invention are especially valuable for the removal of endotoxin which is bound to or associated with proteins, drugs or other biological compounds intended for medical or pharmaceutical applications. Certain biological compounds, particularly proteins, often bind endotoxin tightly and subsequent removal is very difficult, if not impossible, by existing means. Affinity ligand-matrix conjugates of the invention may also be applied to the removal of endotoxin from blood or plasma and so provide an especially useful tool for in vitro or in vivo removal of endotoxin, the latter being achieved, for example, by way of an extracorporeal endotoxin extraction device. Such a device may be especially valuable for removal of endotoxin which is released into the blood stream during bacterial infections, such infections often causing life-threatening diseases such as septicaemia or meningitis. Removal of blood-borne endotoxin may be particularly beneficial in the treatment of these diseases and in the prevention and management of septic shock.
Novel affinity ligand-matrix conjugates provided by this invention can be used in place of other endotoxin binding materials and are significantly more flexible in their use, are more robust, less expensive to produce and offer greater endotoxin binding efficiencies.
The present invention relates to affinity ligand-matrix conjugates comprising a ligand having General Formula (1): 
wherein one of the symbols X represents a nitrogen atom and the other symbol X represents a nitrogen atom or a carbon atom carrying a chlorine atom or a cyano group;
A1 and A2 each independently represent an oxygen atom, a sulphur atom or a group Nxe2x80x94R1;
R1 represents a hydrogen atom, an alkyl group containing from 1 to 6 carbon atoms, a hydroxyalkylgroup containing from 1 to 6 carbon atoms, a benzyl group or a xcex2-phenylethyl group;
B1 and B2 each independently represent an optionally substituted hydrocarbon linkage containing from 1 to 10 carbon atoms (any substituent being substantially non-critical with respect to utility) and including alkyl, phenyl, naphthyl and cyclohexyl groups;
D1 represents a hydrogen atom, a primary amino group, a secondary amino group, a tertiary amino group, a quaternary ammonium group, an imidazole group, a guanidino group or an amidino group;
D2 represents a primary amino group (e.g. as derived from lysine or ornithine), a secondary amino group, a tertiary amino group, a quaternary ammonium group, an imidazole group, a guanidino group or an amidino group; and
p is 0 or 1.
The ligand is attached to a support matrix in position Z, optionally through a spacer arm interposed between the ligand and matrix. Alternatively, in novel ligands of the invention, Z represents a functional group of the type capable of reaction with a solid matrix that may be activated (if necessary or desired) or unactivated.